CN108101975A - A kind of forest mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation method and application - Google Patents
A kind of forest mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of forest mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation methods, belong to Peptide systhesis expression technology field, the amino acid sequence of the polypeptide Sylvsetin is as shown in Seq ID No.1.The present invention also provides the transformation peptide of the polypeptide Sylvestin, the amino acid sequence of the transformation peptide is as shown in Seq ID No.5, Seq ID No.6, Seq ID No.7 or Seq ID No.8.The polypeptide Sylvestin and its transformation peptide have the effect of FXIIa inhibitory activity and antithrombotic and treatment apoplexy.Vivoexpression preparation method of the present invention realizes mountain leech Sylvestin polypeptides and is produced in the in vivo scale of prokaryotes, low cost, by purification application in the preparation of biological function drug.
Description
Technical field
The invention belongs to Peptide systhesis expression technology fields, and in particular to a kind of forest mountain leech antithrombotic polypeptide
Sylvestin and its vivoexpression preparation method and application.
Background technology
Thrombus be cause cardiovascular disease incidence and it is lethal the main reason for, incidence and age, which increase, to be positively correlated.I
State is stepping into the country of aging, and angiocardiopathy becomes a heavy social burden.For a long time, coagulated for blood
Gu, platelet activation and the physiological mechanisms such as aggregation and thrombolysis have developed substantial amounts of antithrombotic reagent.But many anti-blood
Bolt drug because with hemorrhagic activity, cause low blood pressure when systemic side effects due to be restricted.Therefore, excavate new, special
Antithrombotic reagent is the hot spot of research all the time.
In recent years, rat experiment model and clinical research show FXI (plasma thromboplastin precursor) or FXII (contact because
Son) there is important role for the formation of thrombus and stabilization, especially they have a minor impact to hemostasis, i.e. FXI or FXII's
Inhibitor plays an important role of antithrombotic, without having hemorrhage side effect.The FXI or the inhibitor of FXII studied at present includes Dan Ke
There are prices to hold high for grand antibody, antisense oligonucleotides, polypeptide, chemical small molecule etc., wherein monoclonal antibody, antisense oligonucleotides
It is expensive, work slow, the problems such as may being difficult to control, in contrast, polypeptide is more suitable for actual answer as the inhibitor of FXI or FXII
With.And the polypeptide studied at present is there are species is few, activity is low, it is unstable the problem of.
The content of the invention
In view of this, it is an object of the invention to provide a kind of activity is high, stability is good, it is suitable for industrial forest
Mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation method and application.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：A kind of forest mountain leech antithrombotic polypeptide
Sylvestin, the amino acid sequence of the polypeptide Sylvsetin is as shown in Seq IDNo.1.
The present invention provides the nucleotide of the coding polypeptide Sylvestin, the nucleotide sequence such as Seq
Shown in IDNo.2.
The present invention also provides a kind of forest mountain leech antithrombotic recombinant polypeptide Sylvestin, the recombinant polypeptides
Sylvestin includes polypeptide Sylvestin and enterokinase cleavage site segment, and the sequence of the recombinant polypeptide Sylvestin is such as
Shown in Seq IDNo.3.
The present invention provides the nucleotide of the recombinant polypeptide Sylvestin, the nucleotide sequence such as Seq
Shown in IDNo.4.
The present invention also provides a kind of recombinant expression carrier of polypeptide Sylvestin, the expression vector includes such as Seq
Nucleotide sequence shown in IDNo.2.
The present invention provides a kind of recombinant strains of the recombinant expression carrier including polypeptide Sylvestin.
The present invention also provides the transformation peptide that polypeptide Sylvestin rite-directed mutagenesises obtain, the amino acid sequence of the transformation peptide
Row are as shown in Seq IDNo.5, Seq IDNo.6, Seq IDNo.7 or Seq IDNo.8.
The present invention also provides the vivoexpression preparation methods of the polypeptide Sylvestin, comprise the following steps：1) build
Recombinant strains described in claim 6；2) the recombinant bacterial strain expression is induced to obtain the bacterium of the Sylvestin containing recombinant polypeptide
Liquid；The temperature of the induction is 16~30 DEG C, and the IPTG concentration of the induction is 0.05~0.55mmol/L, the induction when
Between be 1~18h；3) isolated and purified from the bacterium solution and obtain recombinant polypeptide Sylvestin；4) restructuring described in enterokinase digestion
Polypeptide Sylvestin obtains polypeptide Sylvestin.
It is anti-in preparation the present invention also provides the forest mountain leech antithrombotic polypeptide Sylvestin or the transformation peptide
Application in thrombus drug.
The present invention also provides the forest mountain leech antithrombotic polypeptide Sylvestin or the transformation peptide to control in preparation
Treat the application in apoplexy drug.
Beneficial effects of the present invention：Polypeptide Sylvestin provided by the present invention is obtained from the separation of forest mountain leech, forest
Mountain leech is a kind of blood sucking annelid, and in order to successfully suck the blood of prey, mountain leech generates in long-term evolutionary process
Many anticoagulant substances resist the solidification of prey blood itself, are anticoagulative substance natural origins.The polypeptide
Sylvestin has FXIIa inhibitory activity, has effects that antithrombotic and curing apoplexy.
Forest mountain leech antithrombotic recombinant polypeptide Sylvestin provided by the present invention is in the sequence of polypeptide Sylvestin
Enterokinase cleavage site is connected, beneficial to connections and polypeptide of the polypeptide Sylvestin in vivoexpression preparation process in carrier
Sylvestin's isolates and purifies.
Transformation peptide provided by the invention is the polypeptide that rite-directed mutagenesis acquisition is carried out on the basis of polypeptide Sylvestin, has
There is the effect of FXIIa inhibitory activity similar with polypeptide Sylvestin and antithrombotic and treatment apoplexy, enrich antithrombotic
The species of polypeptide may be selected in drug and treatment apoplexy drug.
The vivoexpression preparation method of the polypeptide Sylvestin provided by the invention, can prepare high-purity, high
Stability, the homogeneous polypeptide Sylvestin of conformation, the method can enable polypeptide Sylvestin in recombinant strains
Middle high level expression, and the enterokinase in the method cut it is efficient, to polypeptide Sylvestin industrialization productions and its application
It is of great significance.It is raw in the in vivo scale of prokaryotes, low cost that the method realizes mountain leech Sylvestin polypeptides
Production, by purification application in the preparation of biological function drug.
Description of the drawings
Fig. 1 is the PAGE gel electrophoresis of the expression and purification of forest mountain leech Sylvestin recombinant polypeptides Sylvestin
Figure；
The SDS-PAGE that enterokinase is cut under the different condition that Fig. 2 is forest mountain leech antithrombotic recombinant polypeptide Sylvestin coagulates
Gel electrophoresis figure；
Fig. 3 is mass spectral analysis figure after the leech recombinant polypeptide Sylvestin enterokinase digestions of forest mountain；
Fig. 4 cuts rear FXIIa inhibitory activity for forest mountain leech recombinant polypeptide Sylvestin enterokinase；
Fig. 5 cuts rear Sephadex-G50 gel chromatographies figure for forest mountain leech recombinant polypeptide Sylvestin enterokinase；
Fig. 6 is forest mountain leech polypeptide Sylvestin mass spectral analysis figures after purification；
Fig. 7 is forest mountain leech polypeptide Sylvestin with transforming comparative result figure of the peptide to KLK inhibitory activity；
Fig. 8 is forest mountain leech polypeptide Sylvestin with transforming comparative result figure of the peptide to FXIIa inhibitory activity.
Specific embodiment
The present invention provides a kind of forest mountain leech antithrombotic polypeptide Sylvestin, the amino acid of the polypeptide Sylvsetin
Sequence is as shown in SeqIDNo.1, specially：
TSEPVCACPKMLFWVCGKDGETYTHPCIAKCHNVEVEHDGKCK。
Heretofore described polypeptide Sylvestin derives from Yunnan forest mountain leech, and the present invention is excellent in specific implementation process
Being isolated and purified from all homogenates of Yunnan forest mountain leech for choosing obtains polypeptide Sylvestin；The polypeptide Sylvestin can
Extend blood plasma recalcification time.The heretofore described method that polypeptide is isolated and purified from all homogenates of Yunnan forest mountain leech is adopted
With the natural polypeptides isolation and purification method of this field routine, molecular sieve gel is preferably used in specific implementation process
The isolation and purification methods such as filtering, reverse-phase HPLC.
The present invention also provides the nucleotide of the coding polypeptide Sylvestin, the nucleotide sequence such as Seq
Shown in IDNo.2, it is specially：
acctcggaaccggtatgtgcatgcccaaaaatgctattttgggtttgtggtaaagatggtgagacttacacccatcc
ttgcattgcaaaatgccataatgttgaagttgaacatgatgggaagtgcaaataa。
The preparation method of the nucleotide of polypeptide Sylvestin described in heretofore described coding is preferably with the polypeptide
The nucleotides sequence of the amino acid sequence of Sylvestin is classified as template design primer, then from the cDNA library on forest mountain leech head
Middle amplification obtains.Heretofore described specific primer sequence includes forward primer sequence and reverse primer sequences；The forward direction
Primer sequence is specially AAACCGAACCGTCGGTATGT preferably as shown in Seq ID No.13, the reverse primer sequences
It is specially GTTCGGTTTGGTGGCTCATT preferably as shown in Seq ID No.14.
The nucleotide sequence coded polypeptide of heretofore described coded polypeptide Sylvestin is made of 43 amino acid,
Containing six cysteines, molecular weight 4795.4Da is bigger 6Da than the molecular weight of natural polypeptides, this illustrates that natural polypeptides forms
Three pairs of intramolecular disulfide bonds.It compares and finds by BLAST in ncbi database, the polypeptide Sylvestin belongs to Kazal house
The serpin of race.
The present invention, using prokaryotic expression system, recombinates it after the amino acid sequence of the polypeptide Sylvestin is obtained
Albumen is expressed, and after enterokinase is cut, Sylvestin purifying proteins is obtained, through determination of activity APTT (during activated partial fibrin ferment
Between) and PT (thrombin time) is tested, chromophoric substrate detection determines inhibitory activity of the polypeptide Sylvestin to FXIIa.
The present invention provides a kind of forest mountain leech antithrombotic recombinant polypeptide Sylvestin, the recombinant polypeptide Sylvestin
Including polypeptide Sylvestin and enterokinase cleavage site segment, the sequence such as Seq IDNo.3 of the recombinant polypeptide Sylvestin
It is shown, be specially：
MIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSK
GQLKEFLDANLAGSGSGHMHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDD
KAMADIGSTSEPVCACPKMLFWVCGKDGETYTHPCIAKCHNVEVEHDGKCK*。
Heretofore described recombinant polypeptide Sylvestin is the connection enterokinase digestion in the sequence of polypeptide Sylvestin
Site, it is pure in the connection of carrier and the separation of polypeptide Sylvestin beneficial to polypeptide Sylvestin in vivoexpression preparation process
Change.
The present invention provides the nucleotide of the coding recombinant polypeptide Sylvestin, the nucleotide sequence such as Seq
Shown in IDNo.4, it is specially
atgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatca
aaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcgg
caaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggc
catatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaa
attcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatcggatcca
cctcggaaccggtatgtgcatgcccaaaaatgctattttgggtttgtggtaaagatggtgagacttacacccatcct
tgcattgcaaaatgccataatgttgaagttgaacatgatgggaagtgcaaataa。
The present invention also provides a kind of recombinant expression carrier of polypeptide Sylvestin, the expression vector includes such as Seq
Nucleotide sequence shown in IDNo.2.The recombinant expression carrier preferably includes the nucleotide of above-mentioned recombinant polypeptide in the present invention
Sequence；The recombinant expression carrier preferably further includes the expression vector of commercialization, and the expression vector of the commercialization is preferred
Slvestin is preferably purchased from for prokaryotic expression carrier PET32a, the prokaryotic expression carrier PET32a.
The present invention provides a kind of recombinant strains of the recombinant expression carrier including polypeptide Sylvestin.The present invention
The recombinant expression carrier is preferably transformed into expression bacterial strain kind and obtained by the preparation method of the recombinant strains, this
Bacterial strain is expressed described in invention and is preferably BL21 (DE3) pLysS bacterial strains.
The present invention also provides the transformation peptide that polypeptide Sylvestin rite-directed mutagenesises obtain, the amino acid sequence of the transformation peptide
Row are as shown in Seq IDNo.5, Seq IDNo.6, Seq IDNo.7 or Seq IDNo.8.In the present invention to the rite-directed mutagenesis
Method is not particularly limited, using the directed mutagenesis method of this field routine.In the present invention, the core of the transformation peptide is encoded
Nucleotide sequence is as shown in Seq IDNo.9, Seq IDNo.10, Seq IDNo.11 or Seq IDNo.12.It is of the present invention to change
Making peptide has the effect of FXIIa inhibitory activity and antithrombotic similar with polypeptide Sylvestin and treatment apoplexy, enriches
The species of polypeptide may be selected in antithrombotic reagent and treatment apoplexy drug.
The present invention also provides the vivoexpression preparation methods of the polypeptide Sylvestin, comprise the following steps：1) build
Recombinant strains described in claim 6；2) the recombinant bacterial strain expression is induced to obtain the bacterium of the Sylvestin containing recombinant polypeptide
Liquid；The temperature of the induction is 16~30 DEG C, and the IPTG concentration of the induction is 0.05~0.55mmol/L, the induction when
Between be 1~18h；3) isolated and purified from the bacterium solution and obtain recombinant polypeptide Sylvestin；4) restructuring described in enterokinase digestion
Polypeptide Sylvestin obtains polypeptide Sylvestin.
In the present invention, the construction method of the recombinant strains comprises the following steps：A, by enterokinase cleavage site
The nucleotide sequence for obtaining encoding recombinant polypeptide Sylvestin is connected with the nucleotide sequence of coded polypeptide Sylvestin；B, will
The nucleotide sequence of the encoding recombinant polypeptide Sylvestin is connected with expression vector obtains recombinant expression carrier；C, by described in
Recombinant expression carrier is transformed into expression bacterial strain and obtains recombinant strains.
The present invention is by the nucleotide of restriction enzyme site BamH1 and Xhol in plasmid PET32a (+) and coded polypeptide Sylvestin
Sequence connects, and obtains the nucleotide sequence of encoding recombinant polypeptide Sylvestin；The present invention does not have special limit to the connection method
It is fixed, using the nucleotide connection method of this field routine.The present invention is by the enterokinase cleavage site and coded polypeptide
The purpose of the nucleotide sequence connection of Sylvestin is the structure and polypeptide that facilitate subsequent recombinant expression carrier
The purifying of Sylvestin.
The present invention is after the nucleotide sequence of encoding recombinant polypeptide Sylvestin is obtained, by the encoding recombinant polypeptide
The nucleotide sequence of Sylvestin is connected with expression vector, obtains recombinant expression carrier, and the expression vector is preferably protokaryon
Expression vector, more preferably PET32a.The present invention is not particularly limited the method for the connection conventional using this field
Carrier connection kit can be selected in specific implementation process for connection method.
The recombinant expression carrier is transformed into expression bacterial strain and is recombinated after recombinant expression carrier is obtained by the present invention
Express bacterial strain.Heretofore described expression bacterial strain is preferably coli strain, more preferably BL21 (DE3) pLysS bacterium
Strain.
The present invention induces the recombinant bacterial strain expression to obtain containing recombinant polypeptide after recombinant strains are obtained
The bacterium solution of Sylvestin；The temperature of the induction is 16~30 DEG C, preferably 20~29 DEG C, more preferably 28 DEG C；It is described
The IPTG concentration of induction is 0.05~0.55mmol/L, preferably 0.48~0.52mmol/L, more preferably 0.5mmol/
L；The time of the induction is 1~18h, preferably 4~12h, more preferably 8h.
The expansion culture of recombinant strains, heretofore described expansion are preferably further included before heretofore described induction
Culture is specifically that the positive monoclonal of the recombinant strains is inoculated in the LB containing ampicillin (100 μ g/mL) to train
It supports in base, 37 DEG C of shaken cultivations are stayed overnight, and are 1 in inoculum concentration ratio：100 expand culture to bacterium solution OD600It is worth for 0.6~0.7.
The preparation method of the positive monoclonal of heretofore described recombinant strains is using the recombinant strains of this field routine
Positive monocloning method, without other particular/special requirements.
The present invention is isolated and purified from the bacterium solution and recombinated after the bacterium solution of the Sylvestin containing recombinant polypeptide is obtained
Polypeptide Sylvestin.The method for isolating and purifying recombinant polypeptide is preferably：Contain recombinant polypeptide described in collecting
Thalline in the bacterium solution of Sylvestin, the bacterial cell disruption that will be collected into collect the supernatant of the albumen containing recombinant polypeptide, Ran Houjing
Micro-filtration, Ni2+- NTA affinity chromatographys post separation obtains recombinant polypeptide Sylvestin.
The method of the bacterial cell disruption is preferably multigelation method in the present invention, in specific implementation process of the present invention
Combination buffer 15ml is added in the preferred thalline per g, and thalline, multigelation bacterium solution 3 times, Ran Hou under the conditions of -80 DEG C is resuspended
Separation of solid and liquid obtains supernatant after ultrasonication is carried out while ice bath.The combination buffer is preferably wrapped in the present invention
Include 20mmol/L Na3PO4, 0.5mol/L NaCl and 20mmol/L imidazoles aqueous solution；The power of the ultrasound is preferably
600~1000w, more preferably 800w；The ultrasound uses intermittent ultrasound, specially 5s ultrasounds, 5s intervals, the ultrasound
Total time preferred 25~35min, more preferably 30min.
The present invention is after supernatant is obtained, supernatant described in micro-filtration, the micro-filtration with microfiltration membranes be preferably 0.8 μm and
0.45 μm of miillpore filter.The micro-filtration preferably first crosses 0.8 μm of miillpore filter and then after 0.45 μm of miillpore filter.
The present invention is after the supernatant liquid filtering, by obtained micro-filtrate through Ni2+- NTA affinity chromatography post separations are weighed
Group polypeptide Sylvestin；The relative molecular mass of the recombinant polypeptide Sylvestin is about 18.36KDa, meets expected calculating
Value.The heretofore described separated actual conditions of affinity column is preferably：
Wash buffer:20mM Tris-HCl buffer, 100mM NaCl, 10% (v/v) glycerol, 7mM 2-
ME,20mM imidazole,pH8.0.
Elution buffer:20mM Tris-HCl buffer, 100mM NaCl, 10% (v/v) glycerol, 7mM
2-ME,250mM imidazole,pH8.0.
Savingbuffer:20mM Tris-HCl buffer, 100mM NaCl, 10% (v/v) glycerol, 7mM 2-
ME,pH8.0.
The separated specific steps of affinity column are preferably：1. treating that sample supernatant substantially flows only, wash is added in
Buffer washes foreign protein, generally requires the Wash buffer of 10~15 times of bed volumes, and miscellaneous egg is detected with Coomassie G250
It is white whether wash clean.2. such as the non-discolouring expression foreign proteins of Coomassie G250, wash clean (is had to connecing newest outflow
Liquid detecting), 2~3 times of elution buffer elution destination proteins are added in, starts to collect destination protein at this time, be used in combination
Coomassie G250 monitorings are collected.3. the protein solution of collection is transferred in bag filter, bag filter tightens, and places 1~2L
It dialyses in saving buffer albumen.Such as plus stirring, dialysis about 2~3hr are completed.Dialysis need to be in 4 DEG C of completions.4. collect dialysis
Albumen afterwards carries out the purity of SDS-PAGE detection purifying proteins, and measures protein concentration with Bradford methods.5. it carries out
Enzyme activity is tested.
The present invention is after recombinant polypeptide Sylvestin is obtained, and recombinant polypeptide Sylvestin is obtained described in enterokinase digestion
Polypeptide Sylvestin.Heretofore described digestion condition is preferably：Recombinant polypeptide Sylvestin concentration is 1~20mg/ml,
Enzyme amount 0.3~3U/50 μ l, 22~22 DEG C of digestion temperature, digestion time are 14~18h；More preferably protein concentration 10mg/
Ml, enzyme amount be 3U/50 μ l, 21 DEG C of digestion temperature, digestion time 16h.
The present invention preferably further includes the purifying to the polypeptide Sylvestin after the polypeptide Sylvestin is obtained
Process, the purifying of the polypeptide Sylvestin preferably use Sephadex-G50 gel chromatographies and RP-HPLC methods；This hair
Conditional parameter in Sephadex-G50 gel chromatographies described in bright and RP-HPLC procedures uses the condition of this field routine
Parameter.
It is anti-in preparation the present invention also provides the forest mountain leech antithrombotic polypeptide Sylvestin or the transformation peptide
Application in thrombus drug；The forest mountain leech antithrombotic polypeptide Sylvestin or the transformation peptide are as the effective of drug
Dosage is 0.1~20mg/Kg.
The present invention also provides the forest mountain leech antithrombotic polypeptide Sylvestin or the transformation peptide to control in preparation
Treat the application in apoplexy drug.The forest mountain leech antithrombotic polypeptide Sylvestin or transformation peptide the having as drug
Effect dosage is 0.1-20mg/Kg.
With reference to embodiment to a kind of forest mountain leech antithrombotic polypeptide Sylvestin provided by the invention and its external table
It is described in detail up to preparation method and application, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The acquisition of natural forest mountain leech antithrombotic polypeptide Sylvestin
From use molecular sieve gel filtration and reversed-phase high pressure liquid chromatography from all homogenates of Yunnan forest mountain leech
It isolates and purifies and obtains polypeptide Sylvestin.12 amino acid of N-terminal of the polypeptide Sylvestin are determined with Edman edman degradation Edmans
Sequence, and screening obtains encoding the homologous sequence of the polypeptide from the leech transcript profile of Yunnan forest mountain, with the homologous nucleotide sequence
Stencil design specific primer, forward primer AAACCGAACCGTCGGTATGT are classified as, reverse primer sequences are
GTTCGGTTTGGTGGCTCATT.Then amplification has obtained coded polypeptide from the mountain leech head cDNA library of structure
The nucleotide sequence of Sylvestin.
The nucleotide sequence coded mature peptide is made of 43 amino acid, and containing six cysteines, molecular weight is
4795.4Da, bigger 6Da than the molecular weight of natural polypeptides, this illustrates that natural polypeptides forms three pairs of intramolecular disulfide bonds.
Embodiment 2
A kind of forest mountain leech antithrombotic recombinant polypeptide Sylvestin, including polypeptide Sylvestin and enterokinase cleavage site
Segment, sequence are specially as shown in Seq IDNo.3
MKLLVVLLIVSLVGMSHQTSEPVCACPKMLFWVCGKDGETYTHPCIAKCHNVEVEHDGKCK.It is heretofore described heavy
Group polypeptide Sylvestin is to connect enterokinase cleavage site in the sequence of polypeptide Sylvestin, is prepared beneficial to vivoexpression
Polypeptide Sylvestin is in the connection of carrier and isolating and purifying for polypeptide Sylvestin in journey.
Embodiment 3
The transformation peptide that polypeptide Sylvestin rite-directed mutagenesises obtain, amino acid sequence such as Seq IDNo.5 of the transformation peptide,
Shown in Seq IDNo.6, Seq IDNo.7 or Seq IDNo.8.Encode it is described transformation peptide nucleotide sequence such as Seq IDNo.9,
Shown in Seq IDNo.10, SeqIDNo.11 or Seq IDNo.12.
Embodiment 4
A kind of recombinant expression carrier of polypeptide Sylvestin, the recombinant expression carrier are to prokaryotic expression carrier
PET32a is transferred to the nucleotide sequence of recombinant polypeptide Sylvestin.With identical enzyme while digestion prokaryotic expression carrier PET32a
With recombinant polypeptide Sylvestin, the connection i.e. recombinant expression carrier of acquisition peptide Sylvestin after digestion.
Embodiment 5
A kind of recombinant strains of the recombinant expression carrier including polypeptide Sylvestin carry the expression in embodiment 4
Body is transferred in BL21 (DE3) pLysS bacterial strains and obtains.Using heat shock method in the present embodiment, under 42 DEG C of hot shock conditions by described in
Recombinant expression carrier be transferred in BL21 (DE3) pLysS bacterial strains and obtain recombinant strains.
Embodiment 6
The vivoexpression preparation method of polypeptide Sylvestin
Enterokinase cleavage site with the nucleotide sequence of coded polypeptide Sylvestin is connected and obtains encoding recombinant polypeptide
The nucleotide sequence of Sylvestin；The nucleotide sequence of the encoding recombinant polypeptide Sylvestin with expression vector is connected and is obtained
Obtain recombinant expression carrier；
It will verify that correct recombinant expression carrier is transformed into expression bacterial strain BL21 (DE3) pLysS, picking positive monoclonal
It being inoculated in the LB culture mediums containing ampicillin (100 μ g/mL), 37 DEG C of shaken cultivations are stayed overnight, and by 1：100 expand culture extremely
After OD600 values are 0.6~0.7, different Fiber differentiation time cultivation temperatures and IPTG concentration are set respectively, induced
Expression of the Sylvestin in bacterial strain is expressed, it is that temperature is 28 DEG C to obtain optimal expression condition, and IPTG concentration is 0.5mmol/
L, induction time 8h.
Restructuring Sylvestin strains are inoculated into 500ml LB culture mediums (containing 100 μ g/ml ammonia benzyls mycins), 27 DEG C
Under the conditions of after IPTG (0.5mmol/L) inductions recombinant bacterium 8h, 4 DEG C, 12000r/min centrifugations 10min collect thalline, add per g thalline
Enter combination buffer (20mmol/L Na3PO4,0.5mol/L NaCl, 20mmol/L imidazoles) 15ml and thalline is resuspended.At -80 DEG C
Under the conditions of multigelation bacterium solution 3 times, ice bath carries out ultrasonication (800W), and 5s ultrasounds, 5s intervals crush 30min.It is used after broken
4 DEG C, 12000r/min centrifugation 30min collect supernatant and precipitate, proved by PAGE gel electrophoresis, destination protein is
In supernatant, the results are shown in Figure 1, and it is recombinant protein Sylvestin that arrow, which marks, shows that recombinant protein is a large amount of in supernatant
Expression.By supernatant successively with 0.8 μm and 0.45 μm of filtering with microporous membrane, then through Ni2+- NTA affinity chromatographys post separation, purifying mesh
Albumen, obtained purifying Sylvestin recombinant protein relative molecular masses are about 23KDa, and such as Fig. 2 is shown, meets expected meter
Calculation value.
Recombinant polypeptide, digestion condition are cut using enterokinase：Protein concentration is 20,10mg/ml, 1mg polypeptides
Sylvestin, digestion volume are 50 μ l, and it is respectively 3U to add in enzyme amount, and 1U, 0.3U most suitable use enzyme amount to probe into.Digestion temperature
For 21 DEG C, time 16h.Final probe into draws 10mg/ml Sylvestin, and 3U/50 μ l, 21 DEG C, the digestion effect of 16h is most
Good, the results are shown in Figure 2：In the endonuclease reaction system of 50 μ l, the recombinant protein Sylvestin (20mg/ of two concentration are set
Ml, 10mg/ml), add in the enterokinase (3U, 1U, 0.3U) of different unit of activity.It is according to as above 16%SDS-PAGE electrophoretograms
Enterokinase cuts the albumen after His-Sylvestin, is all 15 μ g per hole applied sample amount, it can be seen that when using 10mg/
MlSylvestin, enzyme amount 3U/50ul, 21 DEG C, during 16h, the band of Sylvestin is the most apparent, and it is optimal digestion item to show this
Part.0U is represented by Ni2+After-NTA affinity chromatography column separating purifications, the albumen of enterokinase is not added with.
Albumen after enterokinase digestion, by the albumen after digestion by mass spectral analysis, the results are shown in Figure 3, and arrow is signified
For the peak of the successful Sylvestin albumen of digestion, molecular weight 4789.7 is almost consistent with destination protein molecular size range,
Show digestion success.The FXIIa inhibitory activity of the polypeptide Sylvestin after digestion is measured, the results are shown in Figure 4, after digestion
Sylvestin has enzyme inhibition activity, further demonstrates digestion success, and active.
After digestion, the polypeptide Sylvestin obtained to digestion is purified.Using the super filter tube initial gross separation mesh of 10Kda
Protein S ylvestin with merge head His-tag after, isolate and purify Sylvestin using Sephadex-G50 gel chromatographies, tie
Fruit is as shown in figure 5, arrow is signified for Sylvestin peaks, and (His-sylvestin of 10mg can obtain 1mg to total purifying rate for 10%
Polypeptide Sylvestin).Then the polypeptide Sylvestin that Sephadex-G50 gel chromatographies isolate and purify is passed through into mass spectrum point
Analysis, the results are shown in Figure 6, and molecular weight is consistent with destination protein.
Embodiment 7
The KLK of transformation peptide described in forest mountain leech Sylvestin polypeptides and embodiment 3 described in embodiment 1,
FXIIa inhibitory activity measures
HFXIIa：The FXIIa of final concentration of 0.2 μM of the Sylvestin and 10 μ L of 10 μ L various concentrations is mixed in 96 orifice plates
Together in 40 μ L buffer solutions (100mMNacl, 50mMTris-HCl, pH8.0,5mMCaCl2).It is placed at room temperature for after five minutes, adds in
The mixing of 30 μ L buffer solutions and the final concentration of 0.04mMS2302 of 10 μ L (CHROMOGENIX, H-D-Pro-Arg-pNA2HCl)
Liquid, final volume are 100 μ L.The dynamics of reaction uses Epoch (BioTek) microplate reader, GENCHS1.09 software detections
OD405nm, 20min are spaced 47s.
Plasmakallikrein(KLK)：The Sylvestin of various concentration is mixed with 0.26 μM of KLK in 96 orifice plates
In 40 μ LTris-HCl buffer solutions (20mM, pH7.4).It is placed at room temperature for after five minutes, adds in 30 μ L buffer solutions and 10 μ L color developments bottoms
The mixed liquor of object (Gly-Arg-p-nitroanilidedihydrochloride), final volume are 100 μ L.The dynamics of reaction
Using Epoch (BioTek) microplate reader, GENCHS1.09 software detection OD405nm, 20min, 30s, two repetitions are spaced.
As a result as shown in Figure 7 and Figure 8, the concentration of addition is all the FXIIa of 300 μ g/ml, Sylvestin native peptides,
Kallikrein inhibitory activity highests, therefore can predict that Sylvestin native peptides anticoagulant actives are most strong.
From above-described embodiment, there is FXIIa to inhibit for polypeptide Sylvestin provided by the present invention and transformation peptide
Activity has blood coagulation resisting function, the effect of being provided simultaneously with antithrombotic and treat apoplexy, enriches antithrombotic reagent and treatment apoplexy
The species of polypeptide may be selected in drug.
The vivoexpression preparation method of the polypeptide Sylvestin provided by the invention, can prepare high-purity, high
Stability, the homogeneous polypeptide Sylvestin of conformation, realize mountain leech Sylvestin polypeptides the in vivo scale of prokaryotes,
Low cost production, by purification application in the preparation of biological function drug.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Kunming Institute of Zoology, Chinese Academy of Sciences
<120>A kind of forest mountain leech antithrombotic polypeptide Sylvestin and its vivoexpression preparation method and application
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 43
<212> PRT
<213> Haemadipsa sylvestris
<400> 1
Thr Ser Glu Pro Val Cys Ala Cys Pro Lys Met Leu Phe Trp Val Cys
1 5 10 15
Gly Lys Asp Gly Glu Thr Tyr Thr His Pro Cys Ile Ala Lys Cys His
20 25 30
Asn Val Glu Val Glu His Asp Gly Lys Cys Lys
35 40
<210> 2
<211> 132
<212> DNA
<213> Haemadipsa sylvestris
<400> 2
acctcggaac cggtatgtgc atgcccaaaa atgctatttt gggtttgtgg taaagatggt 60
gagacttaca cccatccttg cattgcaaaa tgccataatg ttgaagttga acatgatggg 120
aagtgcaaat aa 132
<210> 3
<211> 171
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 3
Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys
1 5 10 15
Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro
20 25 30
Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly
35 40 45
Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
50 55 60
Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly His Met His
65 70 75 80
His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly Met
85 90 95
Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser Pro
100 105 110
Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Ala Asp Ile Gly Ser
115 120 125
Thr Ser Glu Pro Val Cys Ala Cys Pro Lys Met Leu Phe Trp Val Cys
130 135 140
Gly Lys Asp Gly Glu Thr Tyr Thr His Pro Cys Ile Ala Lys Cys His
145 150 155 160
Asn Val Glu Val Glu His Asp Gly Lys Cys Lys
165 170
<210> 4
<211> 516
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgatcgccc cgattctgga tgaaatcgct gacgaatatc agggcaaact gaccgttgca 60
aaactgaaca tcgatcaaaa ccctggcact gcgccgaaat atggcatccg tggtatcccg 120
actctgctgc tgttcaaaaa cggtgaagtg gcggcaacca aagtgggtgc actgtctaaa 180
ggtcagttga aagagttcct cgacgctaac ctggccggtt ctggttctgg ccatatgcac 240
catcatcatc atcattcttc tggtctggtg ccacgcggtt ctggtatgaa agaaaccgct 300
gctgctaaat tcgaacgcca gcacatggac agcccagatc tgggtaccga cgacgacgac 360
aaggccatgg ctgatatcgg atccacctcg gaaccggtat gtgcatgccc aaaaatgcta 420
ttttgggttt gtggtaaaga tggtgagact tacacccatc cttgcattgc aaaatgccat 480
aatgttgaag ttgaacatga tgggaagtgc aaataa 516
<210> 5
<211> 43
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 5
Thr Ser Glu Pro Val Cys Ala Cys Pro Lys Met Tyr Phe Trp Val Cys
1 5 10 15
Gly Lys Asp Gly Glu Thr Tyr Thr His Pro Cys Ile Ala Lys Cys His
20 25 30
Asn Val Glu Val Glu His Asp Gly Lys Cys Lys
35 40
<210> 6
<211> 43
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 6
Thr Ser Glu Pro Val Cys Ala Cys Pro Lys Met Leu Val Trp Val Cys
1 5 10 15
Gly Lys Asp Gly Glu Thr Tyr Thr His Pro Cys Ile Ala Lys Cys His
20 25 30
Asn Val Glu Val Glu His Asp Gly Lys Cys Lys
35 40
<210> 7
<211> 43
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 7
Thr Ser Glu Pro Val Cys Ala Cys Pro Lys Met Leu Phe Pro Val Cys
1 5 10 15
Gly Lys Asp Gly Glu Thr Tyr Thr His Pro Cys Ile Ala Lys Cys His
20 25 30
Asn Val Glu Val Glu His Asp Gly Lys Cys Lys
35 40
<210> 8
<211> 43
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 8
Thr Ser Glu Pro Val Cys Ala Cys Phe Arg Asn Leu Phe Trp Val Cys
1 5 10 15
Gly Lys Asp Gly Glu Thr Tyr Thr His Pro Cys Ile Ala Lys Cys His
20 25 30
Asn Val Glu Val Glu His Asp Gly Lys Cys Lys
35 40
<210> 9
<211> 129
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
accagcgagc ccgtgtgcgc ctgccccaag atgtacttct gggtgtgcgg caaggacggc 60
gagacctaca cccacccctg catcgccaag tgccacaacg tggaggtgga gcacgacggc 120
aagtgcaag 129
<210> 10
<211> 129
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
accagcgagc ccgtgtgcgc ctgccccaag atgctggtgt gggtgtgcgg caaggacggc 60
gagacctaca cccacccctg catcgccaag tgccacaacg tggaggtgga gcacgacggc 120
aagtgcaag 129
<210> 11
<211> 129
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
accagcgagc ccgtgtgcgc ctgccccaag atgctgttcc ccgtgtgcgg caaggacggc 60
gagacctaca cccacccctg catcgccaag tgccacaacg tggaggtgga gcacgacggc 120
aagtgcaag 129
<210> 12
<211> 129
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
accagcgagc ccgtgtgcgc ctgcttcagg aacctgttct gggtgtgcgg caaggacggc 60
gagacctaca cccacccctg catcgccaag tgccacaacg tggaggtgga gcacgacggc 120
aagtgcaag 129
<210> 13
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
aaaccgaacc gtcggtatgt 20
<210> 14
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
gttcggtttg gtggctcatt 20
Claims (10)
1. a kind of amino acid sequence such as Seq ID of forest mountain leech antithrombotic polypeptide Sylvestin, the polypeptide Sylvsetin
Shown in No.1.
2. the nucleotide of coding polypeptide Sylvestin described in claim 1, the nucleotide sequence such as Seq ID No.2 institutes
Show.
A kind of 3. forest mountain leech antithrombotic recombinant polypeptide Sylvestin, which is characterized in that the recombinant polypeptide Sylvestin bags
Include polypeptide Sylvestin and enterokinase cleavage site segment, the sequence such as Seq ID No.3 of the recombinant polypeptide Sylvestin
It is shown.
4. encode the nucleotide of the recombinant polypeptide Sylvestin described in claim 3, the nucleotide sequence such as Seq ID
Shown in No.4.
5. a kind of recombinant expression carrier of polypeptide Sylvestin, which is characterized in that the expression vector includes such as Seq ID
Nucleotide sequence shown in No.2.
6. a kind of recombinant strains of polypeptide Sylvestin, which is characterized in that the recombinant strains include claim
Recombinant expression carrier described in 5.
7. the transformation peptide that polypeptide Sylvestin rite-directed mutagenesises described in claim 1 obtain, which is characterized in that the transformation peptide
Amino acid sequence as shown in Seq ID No.5, SeqID No.6, Seq ID No.7 or Seq ID No.8.
8. the vivoexpression preparation method of polypeptide Sylvestin described in claim 1, comprises the following steps：
1) recombinant strains described in claim 6 are built；
2) the recombinant bacterial strain expression is induced, obtains the bacterium solution of the Sylvestin containing recombinant polypeptide；The temperature of the induction for 16~
30 DEG C, the IPTG concentration of the induction is 0.05~0.55mmol/L, and the time of the induction is 1~18h；
3) the recombinant polypeptide Sylvestin for obtaining amino acid sequence shown in Seq ID No.3 is isolated and purified from the bacterium solution；
4) with recombinant polypeptide Sylvestin described in enterokinase digestion, polypeptide Sylvestin is obtained.
9. prepared by forest mountain leech antithrombotic polypeptide Sylvestin described in claim 1 or the transformation peptide described in claim 7
Application in antithrombotic reagent.
10. forest mountain leech antithrombotic polypeptide Sylvestin described in claim 1 or the transformation peptide described in claim 7 are being made
Application in standby treatment apoplexy drug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109260466A (en) * | 2018-06-25 | 2019-01-25 | 邓舒心 | A kind of anticancer drug based on mountain leech |
| CN112759632A (en) * | 2021-02-01 | 2021-05-07 | 昆明龙津药业股份有限公司 | Preparation method of Sylvestin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994009039A1 (en) * | 1992-10-16 | 1994-04-28 | Basf Aktiengesellschaft | New protein that lengthens blood coagulation time, derived from the land leech haemadipsa sylvestris |
| US20090281035A1 (en) * | 2006-06-22 | 2009-11-12 | Novo Nordisk A/S | Soluble Heterodimeric Receptors and Uses Thereof |
| CN108059672A (en) * | 2016-11-08 | 2018-05-22 | 中国科学院昆明动物研究所 | Forest mountain leech antithrombotic peptide Sylvestin and its gene and application |
-
2017
- 2017-12-14 CN CN201711340940.5A patent/CN108101975B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994009039A1 (en) * | 1992-10-16 | 1994-04-28 | Basf Aktiengesellschaft | New protein that lengthens blood coagulation time, derived from the land leech haemadipsa sylvestris |
| US20090281035A1 (en) * | 2006-06-22 | 2009-11-12 | Novo Nordisk A/S | Soluble Heterodimeric Receptors and Uses Thereof |
| CN108059672A (en) * | 2016-11-08 | 2018-05-22 | 中国科学院昆明动物研究所 | Forest mountain leech antithrombotic peptide Sylvestin and its gene and application |
Non-Patent Citations (3)
| Title |
|---|
| DAVID PANTOJA-UCEDA,ET AL.: "Deciphering the Structural Basis That Guides the Oxidative Folding of Leech-derived Tryptase Inhibitor", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 刘伟慧: "森林山蛭（Hameadipsa sylvestris）抗血栓多肽的分离纯化、结构与功能研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 徐小静等: "《生物技术原理与实验》", 31 July 2006, 中央民族大学出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109260466A (en) * | 2018-06-25 | 2019-01-25 | 邓舒心 | A kind of anticancer drug based on mountain leech |
| CN112759632A (en) * | 2021-02-01 | 2021-05-07 | 昆明龙津药业股份有限公司 | Preparation method of Sylvestin |
| CN112759632B (en) * | 2021-02-01 | 2023-08-18 | 昆明龙津药业股份有限公司 | Preparation method of Sylvestin |
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